Nonlinear microscopy (NLM) techniques, such as Two Photon Excited Fluorescence (TPEF) and Second Harmonic Generation (SHG), are able to overcome some of the drawbacks present on conventional confocal laser scanning microscopy (CLSM). This is in part due to the fact that the nonlinear excitation is confined to a focused volume rather than the whole illuminated volume as it is the case for one photon fluorescence. Therefore photo toxicity and out of focus photo bleaching are considerably decreased. This confinement of light is advantageous since it allows optical sectioning of the sample, enabling the reconstruction of three dimensional (3D) models. In addition, nonlinear excitation normally relies on the use of excitation wavelengths in the near-infrared (NIR) range. At these wavelengths besides the fact that there is reduced photo damage, Rayleigh scattering is also decreased enabling larger penetration depths.
A key element in a nonlinear microscope is the use of an ultra-short pulsed (USP) laser. These are natural sources that are able to produce the required high intensities needed for exciting nonlinear processes. Historically, Ti:sapphire sources have been used in NLM due to its available large peak powers along with its large tunabilty range. However, its complexity, high price and maintenance requirements, have limited the widespread adoption of these powerful imaging techniques into “real-life” biomedical applications. Thus, efforts in the past have been concentrated in developing compact, cheap and easy to use USP lasers. However these sources have been limited by the available peak powers. More recently, the use of compact USP laser systems based on Chromium doped gain media such as Cr:LiCAF, Cr:LiSAF, Cr:LiSGAF in NLM and TPEF imaging has been demonstrated. However, these lasers have a limited tuning range constrained by the saturable Bragg reflector design.
Together with those, other alternative sources based on Fiber lasers and semiconductor laser diodes with amplification schemes have also been successfully presented as compact lasers for NLM applications. Fiber lasers can generate very short pulses via passive mode-locking, however, in terms of pulse duration, they can not use the full potential of the gain bandwidth as excessive nonlinearities and higher order chromatic dispersion are present in the fiber. In semiconductor lasers with amplification schemes (i.e. gain-switched laser source based on vertical cavity surface emitting lasers (VCSELs), gain-switched InGaAsP Distributed-Feedback-Bragg (DFB), laser diode and an external cavity mode-locked laser diode consisting of multiple quantum wells (AlGaAs)), the compactness of these systems is hampered by the need to include several stages to compress and/or amplify the pulses.